Fl ovation cdna biotin module v2

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The FL-Ovation cDNA Biotin Module V2 is a component of the Tecan platform designed for the preparation of biotin-labeled cDNA samples. It performs key steps in the cDNA synthesis and labeling workflow to support gene expression analysis applications.

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FL-Ovation Biotin V2 (2 citations) Ovation cDNA module (2 citations) FL-OvationTM cDNA Biotin Module V2 kits (2 citations) FL-Ovation cDNA Biotin Module V2 User Guide (1 citations)

23 protocols using fl ovation cdna biotin module v2

Transcriptome Analysis of Microarray Experiments

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RNA was extracted using Qiagen RNAeasy® Micro Kit, following the manufacturer's
instructions. After RNA extraction, all quantitation and microarray experiments were performed at
the UCLA Department of Pathology Clinical Microarray Core Laboratory. RNA integrity was analyzed
using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). RNA purity and
concentration was determined using a Nanodrop 8000 (Nanodrop Products, Wilmington, DE). Microarray
targets were generated using FL-Ovation cDNA Biotin Module V2 (NuGen Technologies, San Carlos, CA)
and then hybridized to the Affymetrix Gene Chip U133Plus 2.0 Array (Affymetrix, Santa Clara, CA),
all according to the manufacturer's instructions. The arrays were washed and stained with
streptavidin phycoerythrin in Affymetrix GeneChip protocol, and then scanned using an Affymetrix
GeneChip Scanner 3000.

Liu H., Cadaneanu R.M., Lai K., Zhang B., Huo L., An D.S., Li X., Lewis M.S, & Garraway I.P. (2015). Differential Gene Expression Profiling of Functionally and Developmentally Distinct Human Prostate Epithelial Populations. The Prostate, 75(7), 764-776.

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Transcriptome Analysis of Hematopoietic Stem and Progenitor Cells

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ST-HSC (lin⁻ c-kit⁺ Sca-1⁺ CD150⁻ CD48⁻) and GMP (lin⁻ c-kit⁺ Sca-1⁻ CD16/32^hi CD34⁺) were sorted by FACS and RNA was prepared by RNeasy Micro kit (Qiagen). Ten nanograms of total RNA were used for linear amplification of cDNA using the Ovation Pico RNA Amplification System (Nugen), according to the manufacturer’s instructions. Five micrograms of cRNA were biotin-labeled using the FL-Ovation cDNA Biotin Module V2 (NuGen). Following fragmentation, the labeled cRNA of each individual sample was hybridized to Affymetrix MG430 v2 microarrays (Affymetrix) and stained according to the manufacturer’s instructions. Array data have been stored in the gene expression omnibus database (www.ncbi.nlm.nih.gov/geo/; accession no.: GSE27873) according to MIAME standards. Microarray data were normalized using the RMA method with R-Bioconductor, and the differentially expressed genes were identified using the Significance Analysis for Microarrays (SAM) algorithm^{73 (link)} in TM4-MeV software^{74 (link)} with the threshold set to 0 as a median number of falsely significant genes.

Zhao X., Bartholdy B., Yamamoto Y., Evans E.K., Alberich-Jordà M., Staber P.B., Benoukraf T., Zhang P., Zhang J., Trinh B.Q., Crispino J.D., Hoang T., Bassal M.A, & Tenen D.G. (2022). PU.1-c-Jun interaction is crucial for PU.1 function in myeloid development. Communications Biology, 5, 961.

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Transcriptome Profiling of FFPE Tumor Samples

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RNA was extracted from the FFPE tumor samples using RNeasy FFPE kits (Qiagen) and amplified with the WT-Ovation FFPE System V2 (NuGen). cDNA was labeled using the FL-Ovation cDNA Biotin Module V2 (Nugen). Samples were run on the Affymetrix Human Transcriptome Array (HTA) 2.0 following manufacturer’s instructions. For this workflow an Affymetrix Fluidics station 450 and Affymetrix GeneChip Scanner 3000 7G were used. Quality control and normalization was conducted using Affymetrix Expression Console. Of the 134 FFPE samples, 112 samples had sufficient RNA and met quality control criteria (Supplemental figure 1). The microarray data has been deposited in the Gene Expression Omnibus under the accession number GSE76040.

Jeselsohn R., Barry W.T., Migliaccio I., Biagioni C., Zhao J., De Tribolet-Hardy J., Guarducci C., Bonechi M., Laing N., Winer E.P., Brown M., Di Leo A, & Malorni L. (2016). TransCONFIRM: Identification of a genetic signature of response to fulvestrant in advanced hormone receptor positive breast cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(23), 5755-5764.

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Transcriptome analysis of Drosophila hearts

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Drosophila hearts from 1‐ and 5‐week‐old yw wildtype or yw x GMH5 flies (n = 30) were isolated (Fink et al., 2009), removed and homogenized in TRIzol (Invitrogen Life Sciences). Total RNA was precipitated and purified by miRNeasy mini‐column (Qiagen) according to the manufacturer's instructions. The recovered RNA was subjected to first and second strand cDNA synthesis, amplification, and purification using the WT‐ovation Pico RNA Amplification System (NuGEN), Agencourt RNAClean purification beads (Beckman Coulter), and DNA Clean and Concentrator‐25s columns (Zymo Research). cDNA was fragmented and labeled using the FL‐ovation cDNA Biotin Module V2 (NuGEN).

Cannon L., Zambon A.C., Cammarato A., Zhang Z., Vogler G., Munoz M., Taylor E., Cartry J., Bernstein S.I., Melov S, & Bodmer R. (2017). Expression patterns of cardiac aging in Drosophila. Aging Cell, 16(1), 82-92.

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Microarray Data Analysis Pipeline

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Total RNA was extracted and purified using RecoverAllTM Total Nucleic Acid Isolation (Cat#AM1975, Ambion, Austin, TX, US) following the manufacturer’s instructions. Then, the total RNA was amplified, labeled and purified by Affymetrix WT PLUS Reagent Kit (Cat#902280, Affymetrix, Santa Clara, CA, US), Ovation FFPE WTA System (Cat#3403, NuGEN, San Carlos, CA, US) and FL-Ovation™ cDNA Biotin Module V2 (Cat#4200, NuGEN, San Carlos, CA, US). Array hybridization and washing was performed using GeneChip^® Hybridization, Wash and Stain Kit (Cat#900720, Affymetrix, Santa Clara, CA, US) in a Hybridization Oven 645 (Cat#00-0331-220V, Affymetrix, Santa Clara, CA, US) and a Fluidics Station 450 (Cat#00-0079, Affymetrix, Santa Clara, CA, US). Arrays were scanned by Affymetrix GeneChip^® Scanner 3000 (Cat#00-00213, Affymetrix, Santa Clara, CA, US) to obtain raw data. The raw data were normalized by Expression Console software of Affymetrix company. After normalization, the fold changes and p-values of different genes were calculated by the formula: foldchange = average (power (2, signal (FTC)))/average (power (2, signal (BTL))) and t-test separately. Differentially expressed genes (DEGs) were reported if the fold change was >2 or <0.5 and the p-value was smaller than 0.05. At last, Unsupervised consensus clustering was performed by R package “pheatmap” to obtain the heatmap of DEGs.

Yao Y., Xu P., Ying T., Wang Y., Wang X., Shu L., Mo Z., Chen Z., Wang X., Wang W., Teng L, & Lou X. (2022). Integrative Analysis of DNA Methylation and Gene Expression Identified Follicular Thyroid Cancer-Specific Diagnostic Biomarkers. Frontiers in Endocrinology, 12, 736068.

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Bladder Transcriptome Profiling by miRNA & mRNA Microarray

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Agilent Human miRNA Microarray (V3, based on Sanger miRbase release 12.0) was used for measuring miRNA expression in bladder samples. Samples used for the Agilent miRNA microarray were labelled as described by the manufacturer. Affymetrix Human U133 Plus 2.0 arrays (Affymetrix, Santa Clara, CA, USA) were used for measuring gene expression in bladder samples. Samples used for Affymetrix microarray were amplified using the Ovation RNA Amplification System (NuGEN, San Carlos, CA, USA) and labelled using FL-Ovation cDNA Biotin Module V2 (NuGEN) following the manufacturer's protocol. Raw intensity miRNA data were normalised and median transformed using GeneSpring GX 12 (Agilent). The raw mRNA data were log transformed and analysed using Partek Genomics Suite 6.6 (Partek, Saint Louis, MO, USA). Only detected probe sets were used, and compromised or undetected probe sets were filtered out. One-way ANOVA and Tukey's honestly significant difference (HSD) post hoc test were performed across all samples to obtain miRNAs or mRNAs differentially expressed (P<0.05). Unsupervised hierarchical cluster analysis was performed on the list of differentially expressed probe sets with a fold change of ⩾2 (miRNAs) or a fold change of ⩾10 (mRNAs).

Jia A.Y., Castillo-Martin M., Bonal D.M., Sánchez-Carbayo M., Silva J.M, & Cordon-Cardo C. (2014). MicroRNA-126 inhibits invasion in bladder cancer via regulation of ADAM9. British Journal of Cancer, 110(12), 2945-2954.

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Analyzing Liver Gene Expression in Alcohol Consumption

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We completed a comprehensive, microarray analysis of gene expression in liver tissue for 10 patients, each, in the non- and light-alcohol groups. The quality of the isolated ribonucleic acid (RNA) was estimated after electrophoresis using an Agilent 2001 Bioanalyzer (Agilent, Santa Clara, CA). Aliquots (50 ng) of total RNA, isolated from the liver biopsy specimens, were subjected to amplification using the WT-Ovation Pico RNA Amplification System (NuGen, San Carlos, CA), according to the manufacturer's instructions. Approximately 10 μg of complementary deoxyribonucleic acid (cDNA) was amplified from the 50 ng of total RNA, with 5 μg of cDNA used for fragmentation and biotin labeling using the FL-Ovation cDNA Biotin Module V2 (NuGen), according to the manufacturer's instructions. Biotin-labeled cDNA was suspended in 220 μL of a hybridization cocktail (NuGen), with 200 μL of this suspension used for hybridization to the Affymetrix Human 133U Plus 2.0 GeneChip (Affymetrix, Santa Clara, CA) containing 54,675 probes. After stringent washing, the microarray chips were stained with streptavidin-phycoerythrin and probe hybridization was determined using a GeneChip Scanner 3000 (Affymetrix). Data files (CEL) were obtained using the GeneChip Operating Software 1.4 (Affymetrix).

Yamada K., Mizukoshi E., Seike T., Horii R., Kitahara M., Sunagozaka H., Arai K., Yamashita T., Honda M, & Kaneko S. (2018). Light alcohol consumption has the potential to suppress hepatocellular injury and liver fibrosis in non-alcoholic fatty liver disease. PLoS ONE, 13(1), e0191026.

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Total RNA Isolation and Microarray Analysis

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Total RNA was isolated from sorted nucleated Ter119⁺ cells derived from pooled BM of three mice from each genotype. Microarray expression analysis was performed from samples of two independent experiments. Quantity and integrity of the RNA was assessed by nanoelectrophoresis using the Pico Lab-on-a-Chip assay for total eukaryotic RNA using Bioanalyzer 2100 (Agilent Technologies). Only samples with high integrity (RNA integrity number >8) were subsequently used in microarray experiments. Microarray expression profiles were obtained using the Affymetrix GeneChip Mouse Gene 1.0 ST Array (Affymetrix, Santa Clara, CA, USA) and the GCS3000 Affymetrix Platform (Affymetrix). Briefly, 10 ng of total RNA from each sample was amplified using the Ovation Pico WTA System (NuGEN Technologies) and sense transcript cDNA (ST-cDNA) was generated using the WT-Ovation Exon Module (NuGEN Technologies). After, ST-cDNA was fragmented and labeled with the FL-Ovation cDNA Biotin Module V2 (NuGEN Technologies), and the biotinylated cDNA was hybridized to Affymetrix GeneChip Mouse Gene 1.0 ST Arrays. Following hybridization, the array was washed and stained, and finally scanned to generate CEL files for each array. Microarray data have been deposited into the Gene Expression Omnibus (GSE54864).

Farrés J., Llacuna L., Martin-Caballero J., Martínez C., Lozano J.J., Ampurdanés C., López-Contreras A.J., Florensa L., Navarro J., Ottina E., Dantzer F., Schreiber V., Villunger A., Fernández-Capetillo O, & Yélamos J. (2014). PARP-2 sustains erythropoiesis in mice by limiting replicative stress in erythroid progenitors. Cell Death and Differentiation, 22(7), 1144-1157.

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Gene Expression Profiling of mRNAs

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Total RNA (80 ng each sample) was converted to cDNA and amplified using the Applause WT-Amp Plus ST System (NuGEN, San Carlos, CA) and labeled using the FL-Ovation cDNA Biotin Module V2 (NuGEN). The labeled cDNA was then hybridized to Affymetrix GeneChip Human Gene 1.0 ST microarrays (Affymetrix, Santa Clara, CA) to profile gene expression of mRNAs according to the manufacturer protocol. High resolution images of the arrays were obtained and converted to CEL files for analysis.

Liu D.Z., Stamova B., Hu S., Ander B.P., Jickling G.C., Zhan X., Sharp F.R, & Wong B. (2015). MicroRNA and mRNA Expression Changes in Steroid Naïve and Steroid Treated DMD Patients. Journal of Neuromuscular Diseases, 2(4), 387-396.

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Gene Expression Profiling of BRONJ Lesions

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Gene expression profiling was performed using the Affymetrix GeneChip Mouse 430 2.0 Array (Affymetrix, Santa Clara, CA, USA). Total RNA was extracted from osteomucosal samples of BRONJ-like lesions using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instruction. RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and purity/concentration was determined using a NanoDrop 8000 (NanoDrop Products, Wilmington, DE, USA). Microarray experiments were performed at UCLA CMC. Microarray targets were prepared using NuGEN WT-Ovation Formalin-Fixed Paraffin-Embedded RNA Amplification System and FL-Ovation cDNA Biotin Module V2 (NuGEN Technologies, San Carlos, CA, USA) and then hybridized to the array, all according to the manufacturer’s instructions. The arrays were washed and stained with streptavidin phycoerythrin in Affymetrix Fluidics Station 450 using the Affymetrix GeneChip protocol and then scanned using an Affymetrix GeneChip Scanner 3000. The acquisition and initial quantification of array images were conducted using the AGCC software (Affymetrix). The subsequent data analyses were performed using Partek Genomics Suite Version 6.4 (Partek, St. Louis, MO, USA). Differentially expressed genes were selected at ≥2-fold and p< 0.005.

Kim S., Williams D.W., Lee C., Kim T., Arai A., Shi S., Li X., Shin K.H., Kang M.K., Park N.H, & Kim R.H. (2016). IL-36 Induces Bisphosphonate-Related Osteonecrosis of the Jaw-Like Lesions in Mice by Inhibiting TGF-β-Mediated Collagen Expression. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 32(2), 309-318.

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